CN102458387B - New compositions for treating CMT and related disorders - Google Patents

New compositions for treating CMT and related disorders Download PDF

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CN102458387B
CN102458387B CN201080024444.9A CN201080024444A CN102458387B CN 102458387 B CN102458387 B CN 102458387B CN 201080024444 A CN201080024444 A CN 201080024444A CN 102458387 B CN102458387 B CN 102458387B
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CN102458387A (en
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丹尼尔·科亨
谢尔盖·纳比罗钦
伊利亚·楚马科夫
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Pharnext SA
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Abstract

The present invention relates to compositions and methods for the treatment of the Charcot-Marie-Tooth disease and related disorders.

Description

Be used for the treatment of the new compositions of CMT and associated conditions
Technical field
The present invention relates to the compositions and method that are used for the treatment of Sha-Ma-Tu disease (Charcot-Marie-Tooth disease) and associated conditions.
Background technology
Sha-Ma-Tu disease (" CMT ") is the multiple orphan's neuropathy in a kind of heritability periphery.Have 1 people to suffer from this disease in about 2500 individualities, it is the modal hereditary conditions of peripheral nervous system.Its outbreak typically appeared at first or second of life in 10 years, although also can detect it at infancy stage.Lysis is chronic, has neuromuscular degeneration gradually.Be attended by neuralgia and the amyasthenic case of extreme, disease can cause maimed person.CMT is one of heredopathia studying the most deep, employs about 30 in France, 000 case.Although most of CMT patient, with the repetition (CMT1A form) of No. 17 chromosome segments containing myelin gene PMP22, has the gene of more than two-combats relevant to multi-form CMT.Therefore, although origin is monogenic, due to possible regulator gene, this disease indicia goes out Clinical heterogeneity.The gene suddenlyd change in CMT patient, bunch collection is around the interactional molecular pathways affecting Schwann cell or neuronic differentiation or change these cells in peripheral nervous of tight association.
Excavate public data, molecular mechanism and the pathological manifestations of CMT1A disease are described, allow us to several functional cell assembly---the transcriptional control of PMP22 gene, PMP22 albumen folding/degraded, the proliferation and apoptosis of Schwann cell, neuronal death, extracellular matrix deposition and reinvent, immunne response---as the potential rational target that CMT associated treatment is intervened, arrange by priority.The functional assembly of these imbalance controls to the generation of pathological manifestations of Sha-Ma-Tu disease and the combined effect of development, for the latent effectiveness of combination CMT therapy provides proof.
International patent application n ° PCT/EP2008/066457 describes dynamic model by setting up pathology and the targeting functional cell approach relevant to the regulation and control of CMT disease, identifies the method for the drug candidates for the treatment of Sha-Ma-Tu disease.
International patent application n ° PCT/EP2008/066468 describes the compositions being used for the treatment of Sha-Ma-Tu disease, and it comprises at least two kinds of compounds being selected from multi-medicament material standed for.
Summary of the invention
The object of this invention is to provide the new healing potion combination being used for the treatment of CMT and associated conditions.Therefore, the present invention relates to the compositions using certain drug combined therapy CMT and associated conditions, particularly toxic neuropathy and amyotrophic lateral sclerosis and method.
More particularly, one object of the present invention relates to the compound, their salt or the prodrug that comprise baclofen, Sorbitol and be selected from pilocarpine, thiamazole, mifepristone, naltrexone, rapamycin, flurbiprofen and ketoprofen, for simultaneously, delivers medicine to mammalian object respectively or in succession.
Specific purposes of the present invention relate to the compositions comprising baclofen, Sorbitol and naltrexone, and it is for simultaneously, deliver medicine to mammalian object respectively or in succession.
Another object of the present invention relates to the compositions comprising (a) rapamycin, (b) mifepristone or naltrexone and (c) PMP22 regulator, and it is for simultaneously, deliver medicine to mammalian object respectively or in succession.
In a particular embodiment, PMP22 regulator is selected from acetazolamide, albuterol, amiloride, aminoglutethimide, amiodarone, aztreonam, baclofen, balsalazide, betanin, bethanechol, bicalutamide, bromocriptine, bumetanide, buspirone, carbachol, carbamazepine, carbimazole, cevimeline, ciprofloxacin, clonidine, curcumin, cyclosporin A, diazepam, diclofenac, dinoprostone, disulfiram, D-Sorbitol, dutasteride, estradiol, exemestane, felbamate, fenofibrate, finasteride, flumazenil, flunitrazepam, flurbiprofen, furosemide, gabapentin, galantamine, haloperidol, ibuprofen, isoproterenol, ketoconazole, ketoprofen, L-BETAIN, liothyronine (T3), lithium, losartan, loxapine, meloxicam, alotec, metaradrine, metformin, methacholine chloride, thiamazole, D-lysergic acid (+)-butanolamide-(2), metoprolol, metyrapone, miconazole, mifepristone, nadolol, naloxone, naltrexone, norfloxacin, pentazocine, phenoxybenzamine, phenyl butyrate, pilocarpine, pioglitazone, prazosin, propylthiouracil, raloxifene, rapamycin, rifampicin, simvastatin, spironolactone, tacrolimus, tamoxifen, trehalose, trilostane, valproic acid and salt thereof or prodrug.
Another object of the present invention is the compositions comprising rapamycin and mifepristone, and it is for simultaneously, deliver medicine to mammalian object respectively or in succession.
Another object of the present invention is compositions as disclosed above, and it also comprises one or more pharmaceutically acceptable excipient or carrier (i.e. pharmaceutical composition).
Another object of the present invention relates to compositions as disclosed above, and it is used for the treatment of CMT or associated conditions.
Another object of the present invention relates to the combination of compound disclosed in use as above, for the manufacture of the medicine for the treatment of CMT or associated conditions.
Another object of the present invention is the method being used for the treatment of CMT or associated conditions, and described method comprises the compositions as defined above of the object effective dosage to needs.
Another object of the present invention is the method for pharmaceutical compositions, and described method comprises and is blended in applicable excipient or carrier by above-claimed cpd.
Another specific purposes of the present invention are the methods for the treatment of CMT1a in object, and described method comprises as above disclosed compound to the object effective dosage of needs or compound combination.
Another specific purposes of the present invention are the methods for the treatment of toxic neuropathy in object, and described method comprises as above disclosed compound to the object effective dosage of needs or compound combination.
Another specific purposes of the present invention are methods of ALS in treatment in object, described method comprise to needs object effective dosage as above disclosed in compound or compound combination.
Any one in various application disclosed herein or Therapeutic Method, can also comprise by patient diagnosis for suffering from CMT or associated conditions particularly CMT1A, or is have the optional step that CMT or the associated conditions particularly risk of CMT1A occurs by Individual identification.
Thus, another object of the present invention is the method for the treatment of CMT, particularly CMT1a, described method comprises (1) evaluation object and whether suffers from CMT, particularly CMT1a, and (2) suffer from the object of CMT, particularly CMT1a with the combined therapy of the above-claimed cpd of effective dose.Determine whether object suffers from CMT, particularly CMT1a, can be undertaken by the art known various test cases such as DNA algoscopy own.
The present invention is used in any mammalian object, particularly human subjects and treats CMT or associated conditions, is more preferably CMT1a.
Accompanying drawing explanation
Fig. 1. the cooperative effect of drug regimen, dosage 1:A) mixture 7 (dosage 1,10th day), B) d-Sorbitol (SRB, 500 μMs, 10th day), C) (R/S)-baclofen (BCL, 5 μMs, the 10th day) and D) naltrexone (NTX, 5 μMs, the 10th day) impact that MBP is expressed.*: p < 0.05: with contrast (=ascorbic acid) significant difference (then unidirectional ANOVA carries out Fisher post-hoc tests); Ns: no difference of science of statistics
Fig. 2. the cooperative effect of drug regimen, dosage 6:A) mixture 7 (dosage 6,10th day), B) SRB (160 μMs, 10th day), C) BCL (1.6nM, the 10th day) and D) NTX (1.6nM, the 10th day) impact that MBP is expressed.*: p < 0.05: with contrast (=ascorbic acid) significant difference (then unidirectional ANOVA carries out Fisher post-hoc tests); Ns: no difference of science of statistics
Fig. 3. with the A of ascorbic acid altogether incubation in PMP22 TG coculture) the 10th day and B) the 11st day, the positive-effect that mixture 7 (7 kinds of dosage) is expressed MBP, represents with the percentage rate contrasting (=ascorbic acid).Then unidirectional Anova carries out Fisher post-hoc tests.
Fig. 4. what use bar test to measure processes the impact on male rat in 3 and 6 weeks with mixture 1.Waiting time, (white bars represented the control rats by placebo treatment by the meansigma methods tolerance of front twice mensuration of test; Black bar represents the transgenic rat by placebo treatment; Grey bar represents the transgenic rat processed with mixture 1.**p＜0.01。Statistics uses Student two-way test to realize).
Fig. 5. with mixture 1 compositions-treated 3 and 6 weeks (being respectively left figure and right figure), to the positive-effect of male rat gait, (white bars represents smooth gait; Grey bar represents not smooth gait; Black bar represents that rat has serious walking anergy.Statistics uses Student two-way test to realize).
Fig. 6. use surface test (25 °), in rat, mixture 1 compositions is to the positive-effect of male rat.After 3,6,9 and 12 weeks, rat is checked that (white bars represents the control rats by placebo treatment in process; Black bar represents the transgenic rat by placebo treatment; Grey bar represents the transgenic rat processed with mixture 1.*p＜0.05。Statistics uses Student two-way test to realize).
Fig. 7. use surface test (25 °), by mixture 2 compositions-treated, after 3 weeks, to the positive-effect of female rats, (white bars represents the control rats by placebo treatment in rats; Black bar represents the transgenic rat by placebo treatment; Grey bar represents the transgenic rat processed with mixture 2.**p＜0.01。Statistics uses Student two-way test to realize).
Fig. 8. after the neuropathy of oxaliplatin induction, (white bars represents the wild-type rats by placebo treatment to the protective effect of mixture 1 pair of male rat; Black bar represents the wild-type rats with the process of Reference Product gabapentin; Grey bar represents the wild-type rats processed with mixture 1.*p＜0.05；**p＜0.01。Statistics uses Student two-way test to realize).
Fig. 9. observe after processing 9 weeks with mixture 7-dosage 3 in the transgenic animal of process compared with PMP22 transgenic rat the remarkable reduction of pmp22 rna expression (MPZ is as reference gene, Sereda etc., 1996) (p=0.0015).The process LAN of genetically modified integration and pmp22 gene is also proved; In transgenic PMP22 rat pmp22 RNA contrast with its wild type litter newborn animal compared with process LAN 1.8 times (p < 1.10-4).The sciatic nerve of the male rat in 16 week age carries out pmp22 RNA extraction (for n=18 wild type, for n=20 transgenic rat, for n=18 the TG using mixture 7-dosage 3 to process).Statistical analysis uses Welch t-inspection to carry out.
Figure 10. cluster analysis is carried out to the surface test score value under 35 °, to be assigned to when the time point of all assessments the bad, medium of (process 3,6 was analyzed together with 9 weeks) and to perfect performance classification.Significant difference is observed: the WT of 68% belongs to good behaviour group, and only has the TG placebo of 5% to belong to this group (p=0.0003) between WT and TG placebo.Mixture 7-dosage 2 and dosage 3 improve the performance of TG rat.By carrying out statistical analysis (for n=18 WT placebo rat with 5% significance level application trend inspection, for n=20 TG placebo rat, for mixture 7-dosage 2 process TG n=17, for mixture 7-dosage 3 process TG n=18).
Figure 11. use and adopt the Cox model analysis of sandwich variance evaluation mixture 7-dosage 3 to process TG rat after 9 weeks to fall the waiting time in bar is tested, and by comparing with the TG placebo group of 5% significance level use sequence check and reference.What after process 9 weeks, mixture 7-dosage 3 significantly increased TG rat falls the waiting time.
Figure 12. in all time range after treatment (3,6 and 9 weeks), use adopt the Cox model of sandwich variance evaluation to wild type group, transgenic placebo group and with process mixture 7-dosage 3 every day the grip strength of transgenic animal group of totally 9 weeks carry out modeling, and by comparing with the TG placebo group of 5% significance level use sequence check and reference.Corresponding p-value is presented on Kaplan-Meier curve.Observed transgenic placebo rat (melanin line, n=21) and WT rat (grey pigmented line, p=1.45.10 -5, n=19) and compare the remarkable reduction of fore paw grip strength.The strength (black dotted lines, p=0.03, n=18) of fore paw is significantly increased with the process of mixture 7-dosage 3.
The significant correlation fallen between waiting time (after process 9 weeks) and pmp22 rna expression level in the test of Figure 13 .Pearson correlation test display rod: p=1.6.10 -4(analyzing together with the TG that WT, TG placebo and mixture 7-dosage 3 process); P=0.07 (analyzing together with the TG that TG placebo and mixture 7-dosage 3 process).Pmp22 rna expression is lower, and bar test performance is better.Male rat be 16 week age (for n=18 WT rat, white circle; For n=20 TG placebo, black circles; For mixture 7-dosage 3 process TG n=18, white triangles shape).
The significant correlation fallen between waiting time (after process 9 weeks) and the conduction velocity (NCV) of Sensitive nerve in the test of Figure 14 .Pearson correlation test display rod: p=1.34.10 -6(analyzing together with the TG that WT, TG placebo and mixture 7-dosage 3 process); P=0.04 (analyzing together with the TG that TG placebo and mixture 7-dosage 3 process).Conduction velocity is higher, and the performance in bar test is better.Male rat be 16 week age (for n=18 WT rat, white circle; For n=20 TG placebo, black circles; For mixture 7-dosage 3 process TG n=18, white triangles shape).
Detailed Description Of The Invention
The invention provides the new Therapeutic Method being used for the treatment of CMT or associated conditions.The invention discloses new drug regimen, it allows effective rectification of this disease and may be used for any mammalian object.
In background of the present invention, CMT comprises CMT1A, CMT1B, CMT1C, CMT1D, CMT1X, CMT2A, CMT2B, CMT2D, CMT2E, CMT2-P0, CMT4A, CMT4B1, CMT4B2, CMT4D, CMT4F, CMT4 or AR-CMT2A, is more preferably CMT1a.
In background of the present invention, term " CMT associated conditions " refers to and forms the relevant other diseases of the PMP22 unconventionality expression of exception and neuron loss to causing myelin.More particularly, " CMT associated conditions " refers to A Cihai Mo's disease (AD), AD type senile dementia (SDAT), Parkinson's disease, dementia with Lewy body, vascular dementia, autism, mild cognitive disease (MCI), increase rheological properties memory disorders (AAMI) and the problem relevant to aging, Parkinson's disease after encephalitis, schizophrenia, depression, bipolar disorder and other emotional conditions, Huntington chorea, motor neuron disease comprises amyotrophic lateral sclerosis (ALS), multiple sclerosis, idiopathic neuropathies, diabetic neuropathy, toxic neuropathy comprises the neuropathy of being induced by Drug therapy, by HIV, radiation, the neuropathy that heavy metal and vitamin deficiency state cause, based on the neural degeneration of Protein virus, comprise Creutzfeldt-Jakob disease (CJD), mad cow disease (BSE), GSS, FFI, Kuru disease and Alper ' s syndrome.
In preferred embodiments, " CMT associated conditions " refers to toxic neuropathy, particularly drug-induced neuropathy, or ALS.
When using in this article, " treatment " of disease comprises treatment, prevents, prevents, blocks or alleviate the pain caused by disease.Term treatment is particularly including control advancing of disease and related symptoms.
In addition, term compound refers to the chemical compound of concrete name in this application, and any pharmaceutical composition with its acceptable salt, hydrate, ester, ether, isomer, raceme, conjugate and prodrug.The compound listed in the application also can with its corresponding No. CAS identification.
Therefore, the preferred compound used in the present invention is baclofen (CAS 1134-47-0) and possible salt, enantiomer, raceme, prodrug and derivant; Sorbitol (CAS50-70-4) and possible salt, enantiomer, raceme, prodrug and derivant; Naltrexone (CAS 16590-41-3) and possible salt, enantiomer, raceme, prodrug and derivant; Mifepristone (CAS 84371-65-3) and possible salt, enantiomer, raceme, prodrug and derivant; Pilocarpine (CAS 54-71-7) and possible salt, enantiomer, raceme, prodrug and derivant; Thiamazole (CAS 60-56-0) and possible salt, enantiomer, raceme, prodrug and derivant; Ketoprofen (CAS22071-15-4) and possible salt, enantiomer, raceme, prodrug and derivant; Flurbiprofen (5104-49-4) and possible salt, enantiomer, raceme, prodrug and derivant, and rapamycin (CAS 53123-88-9) and possible salt, enantiomer, raceme, prodrug and derivant.
Other compounds used in the present invention are acetazolamide (CAS 59-66-5) and possible salt, enantiomer, prodrug and derivant; Albuterol (CAS 18559-94-9) and possible salt, enantiomer, prodrug and derivant; Amiloride (CAS2016-88-8) and possible salt, enantiomer, prodrug and derivant; Aminoglutethimide (CAS 125-84-8) and possible salt, enantiomer, prodrug and derivant; Amiodarone (CAS 1951-25-3) and possible salt, enantiomer, prodrug and derivant; Aztreonam (CAS 78110-38-0) and possible salt, enantiomer, prodrug and derivant; Baclofen (CAS 1134-47-0) and possible salt, enantiomer, prodrug and derivant; Balsalazide (CAS 80573-04-2) and possible salt, enantiomer, prodrug and derivant; Betanin (CAS 107-43-7) and possible salt, enantiomer, prodrug and derivant; Bethanechol (CAS 674-38-4) and possible salt, enantiomer, prodrug and derivant; Bicalutamide (CAS 90357-06-5) and possible salt, enantiomer, prodrug and derivant; Bromocriptine (CAS25614-03-3) and possible salt, enantiomer, prodrug and derivant; Bumetanide (CAS 28395-03-1) and possible salt, enantiomer, prodrug and derivant; Buspirone (CAS 36505-84-7) and possible salt, enantiomer, prodrug and derivant; Carbachol (CAS 51-83-2) and possible salt, enantiomer, prodrug and derivant; Carbamazepine (CAS 298-46-4) and possible salt, enantiomer, prodrug and derivant; Carbimazole (CAS 22232-54-8) and possible salt, enantiomer, prodrug and derivant; Cevimeline (CAS 107233-08-9) and possible salt, enantiomer, prodrug and derivant; Ciprofloxacin (CAS 85721-33-1) and possible salt, enantiomer, prodrug and derivant; Clonidine (CAS4205-90-7) and possible salt, enantiomer, prodrug and derivant; Curcumin (CAS458-37-7) and possible salt, enantiomer, prodrug and derivant; Cyclosporin A (CAS 59865-13-3) and possible salt, enantiomer, prodrug and derivant; Diazepam (CAS 439-14-5) and possible salt, enantiomer, prodrug and derivant; Diclofenac (CAS 15307-86-5) and possible salt, enantiomer, prodrug and derivant; Dinoprostone (CAS 363-24-6) and possible salt, enantiomer, prodrug and derivant; Disulfiram (CAS 97-77-8) and possible salt, enantiomer, prodrug and derivant; D-Sorbitol (CAS 50-70-4) and possible salt, enantiomer, prodrug and derivant; Dutasteride (CAS 164656-23-9) and possible salt, enantiomer, prodrug and derivant; Estradiol (CAS 50-28-2) and possible salt, enantiomer, prodrug and derivant; Exemestane (CAS107868-30-4) and possible salt, enantiomer, prodrug and derivant; Felbamate (CAS 25451-15-4) and possible salt, enantiomer, prodrug and derivant; Fenofibrate (CAS 49562-28-9) and possible salt, enantiomer, prodrug and derivant; Finasteride (CAS 98319-26-7) and possible salt, enantiomer, prodrug and derivant; Flumazenil (CAS 78755-81-4) and possible salt, enantiomer, prodrug and derivant; Flunitrazepam (CAS 1622-62-4) and possible salt, enantiomer, prodrug and derivant; Flurbiprofen (CAS 5104-49-4) and possible salt, enantiomer, prodrug and derivant; Furosemide (CAS 54-31-9) and possible salt, enantiomer, prodrug and derivant; Gabapentin (CAS60142-96-3) and possible salt, enantiomer, prodrug and derivant; Galantamine (CAS 357-70-0) and possible salt, enantiomer, prodrug and derivant; Haloperidol (CAS 52-86-8) and possible salt, enantiomer, prodrug and derivant; Ibuprofen (CAS 15687-27-1) and possible salt, enantiomer, prodrug and derivant; Isoproterenol (CAS 7683-59-2) and possible salt, enantiomer, prodrug and derivant; Ketoconazole (CAS 65277-42-1) and possible salt, enantiomer, prodrug and derivant; Ketoprofen (CAS 22071-15-4) and possible salt, enantiomer, prodrug and derivant; L-BETAIN (CAS 541-15-1) and possible salt, enantiomer, prodrug and derivant; Liothyronine (T3) (CAS6893-02-3) and possible salt, enantiomer, prodrug and derivant; Lithium (CAS7439-93-2) and possible salt, enantiomer, prodrug and derivant; Losartan (CAS114798-26-4) and possible salt, enantiomer, prodrug and derivant; Loxapine (CAS 1977-10-2) and possible salt, enantiomer, prodrug and derivant; Meloxicam (CAS 71125-38-7) and possible salt, enantiomer, prodrug and derivant; Alotec (CAS 586-06-1) and possible salt, enantiomer, prodrug and derivant; Metaradrine (CAS 54-49-9) and possible salt, enantiomer, prodrug and derivant; Metformin (CAS 657-24-9) and possible salt, enantiomer, prodrug and derivant; Methacholine chloride (CAS 55-92-5) and possible salt, enantiomer, prodrug and derivant; Thiamazole (CAS 60-56-0) and possible salt, enantiomer, prodrug and derivant; D-lysergic acid (+)-butanolamide-(2) (CAS 113-42-8) and possible salt, enantiomer, prodrug and derivant; Metoprolol (CAS37350-58-6) and possible salt, enantiomer, prodrug and derivant; Metyrapone (CAS 54-36-4) and possible salt, enantiomer, prodrug and derivant; Miconazole (CAS 22916-47-8) and possible salt, enantiomer, prodrug and derivant; Mifepristone (CAS 84371-65-3) and possible salt, enantiomer, prodrug and derivant; Nadolol (CAS 42200-33-9) and possible salt, enantiomer, prodrug and derivant; Naloxone (CAS 465-65-6) and possible salt, enantiomer, prodrug and derivant; Naltrexone (CAS 16590-41-3) and possible salt, enantiomer, prodrug and derivant; Norfloxacin (CAS 70458-96-7) and possible salt, enantiomer, prodrug and derivant; Pentazocine (CAS 359-83-1) and possible salt, enantiomer, prodrug and derivant; Phenoxybenzamine (CAS59-96-1) and possible salt, enantiomer, prodrug and derivant; Phenyl butyrate (CAS1821-12-1) and possible salt, enantiomer, prodrug and derivant; Pilocarpine (CAS 54-71-7) and possible salt, enantiomer, prodrug and derivant; Pioglitazone (CAS 111025-46-8) and possible salt, enantiomer, prodrug and derivant; Prazosin (CAS 19216-56-9) and possible salt, enantiomer, prodrug and derivant; Propylthiouracil (CAS 51-52-5) and possible salt, enantiomer, prodrug and derivant; Raloxifene (CAS 84449-90-1) and possible salt, enantiomer, prodrug and derivant; Rapamycin (CAS 53123-88-9) and possible salt, enantiomer, prodrug and derivant; Rifampicin (CAS 13292-46-1) and possible salt, enantiomer, prodrug and derivant; Simvastatin (CAS79902-63-9) and possible salt, enantiomer, prodrug and derivant; Spironolactone (CAS 52-01-7) and possible salt, enantiomer, prodrug and derivant; Tacrolimus (CAS 104987-11-3) and possible salt, enantiomer, prodrug and derivant; Tamoxifen (CAS 10540-29-1) and possible salt, enantiomer, prodrug and derivant; Trehalose (CAS 99-20-7) and possible salt, enantiomer, prodrug and derivant; Trilostane (CAS 13647-35-3) and possible salt, enantiomer, prodrug and derivant; Valproic acid (CAS 99-66-1) and possible salt, enantiomer, prodrug and derivant.
Term " combination " to refer to several drugs co-administered in object to cause the processing method of biological effect.In combination treatment, medicine can together or separately, simultaneously or administration in succession.In addition, medicine also can by different approach or administration.
Now, the present invention is disclosed as qualification and the activity that CMT provides the concrete drug regimen of effectively treatment.More particularly, the invention discloses new triple combination, it provides the appreciable impact on CMT or associated conditions in vitro and in vivo.
Thus, the present invention relates to compositions, its compound comprising baclofen, Sorbitol and be selected from pilocarpine, thiamazole, mifepristone, naltrexone, rapamycin, flurbiprofen and ketoprofen, and their salt, enantiomer, raceme or prodrug.
More particularly, the present invention relates to compositions, its compound comprising baclofen, Sorbitol and be selected from pilocarpine, thiamazole, mifepristone, naltrexone and ketoprofen.
In the most preferred embodiment, the present invention relates to the compositions comprising naltrexone, baclofen and Sorbitol, it is for simultaneously, deliver medicine to mammalian object respectively or in succession.
Under preferable case, in above-mentioned composition, Sorbitol is D-Sorbitol, and baclofen is RS-baclofen or S(-)-Baclofen, is more preferably RS-baclofen.
Another selected objective target of the present invention relates to compositions, and it comprises:
(a) rapamycin,
(b) mifepristone or naltrexone, and
(c) PMP22 regulator,
For simultaneously, deliver medicine to mammalian object respectively or in succession.
Another preferred object of the present invention is compositions, and it comprises:
(a) rapamycin,
(b) mifepristone, and
(c) PMP22 regulator,
For simultaneously, deliver medicine to mammalian object respectively or in succession.
PMP22 regulator can be any compound regulating PMP22 approach in cell and cause or contribute to the normalization of myelin tissue and/or the suppression of neuron loss in itself.PMP22 regulator can be selected from acetazolamide, albuterol, amiloride, aminoglutethimide, amiodarone, aztreonam, baclofen, balsalazide, betanin, bethanechol, bicalutamide, bromocriptine, bumetanide, buspirone, carbachol, carbamazepine, carbimazole, cevimeline, ciprofloxacin, clonidine, curcumin, cyclosporin A, diazepam, diclofenac, dinoprostone, disulfiram, D-Sorbitol, dutasteride, estradiol, exemestane, felbamate, fenofibrate, finasteride, flumazenil, flunitrazepam, flurbiprofen, furosemide, gabapentin, galantamine, haloperidol, ibuprofen, isoproterenol, ketoconazole, ketoprofen, L-BETAIN, liothyronine (T3), lithium, losartan, loxapine, meloxicam, alotec, metaradrine, metformin, methacholine chloride, thiamazole, D-lysergic acid (+)-butanolamide-(2), metoprolol, metyrapone, miconazole, mifepristone, nadolol, naloxone, naltrexone, norfloxacin, pentazocine, phenoxybenzamine, phenyl butyrate, pilocarpine, pioglitazone, prazosin, propylthiouracil, raloxifene, rapamycin, rifampicin, simvastatin, spironolactone, tacrolimus, tamoxifen, trehalose, trilostane, valproic acid, and salt or prodrug.
In preferred embodiments, compound (c) is selected from pilocarpine, thiamazole and baclofen.Thus, most preferred group compound of the present invention comprises:
(a) rapamycin,
(b) mifepristone, and
C () is selected from the compound of pilocarpine, thiamazole and baclofen,
For simultaneously, deliver medicine to mammalian object respectively or in succession.
The instantiation of this compositions comprises the compositions comprising following component:
-rapamycin, mifepristone and pilocarpine;
-rapamycin, mifepristone and baclofen;
-rapamycin, mifepristone and thiamazole; Or
-rapamycin, naltrexone and thiamazole.
Experimental section shows, and the combination of these certain drug effectively can be corrected PMP22 in vitro and be expressed, and is formed and Neuronal integrity, and therefore improve CMT in animal body to recover normal myelin.Result also shows, and these combinations can watch for animals and avoid the neuropathy of chemotherapy induction.Therefore, these compositionss can be used for the neuropathy stoping or alleviate chemotherapy induction, thus enable patient accept the chemotherapy of longer-term.
Another object of the present invention is compositions, and it comprises naltrexone, baclofen and other different PMP22 inhibitor as defined above.
Another object of the present invention is as above disclosed compositions, and it also comprises one or more pharmaceutically acceptable excipient or carrier (i.e. pharmaceutical composition).
Another object of the present invention relates to as above disclosed compositions, and it is used for the treatment of CMT or associated conditions.
Another object of the present invention relates to the application of as above disclosed compound combination, for the manufacture of the medicine for the treatment of CMT or associated conditions.
Another object of the present invention is the method being used for the treatment of CMT or associated conditions, and described method comprises the compositions as defined above of the object effective dosage to needs.
Another object of the present invention is the method for pharmaceutical compositions, and described method comprises and is blended in applicable excipient or carrier by above-claimed cpd.
Of the present invention one more specifically object be the method for the treatment of CMT1a in object, described method comprises as above disclosed compound to the object effective dosage of needs or compound combination.
Another specific purposes of the present invention are the methods for the treatment of toxic neuropathy in object, and described method comprises as above disclosed compound to the object effective dosage of needs or compound combination.
Another specific purposes of the present invention are the methods for the treatment of ALS in object, and described method comprises as above disclosed compound to the object effective dosage of needs or compound combination.
Therapy of the present invention can be carried out as drug regimen, and/or combines with any other therapy and carry out.It can be in, doctor's office, clinic, hospital clinic or hospital provide, so that doctor can the effect of close observation therapy, and makes the adjustment of any needs.
Persistent period of therapy depend on the stage of institute's disease therapy, the age of patient and situation and patient for treatment response how.
In addition, there is the higher people of the risk of other neuropathic conditions (such as to such as diabetes, there is genetic predisposition or suffer from the people of diabetes, or carry out the people etc. for the treatment of of neoplastic conditions), can prophylactic treatment be accepted, to alleviate or to postpone final neuropathic response.
In combination, the dosage of often kind of component, frequency and mode can control independently.Such as, a kind of medicine can be oral, and the second medicine can intramuscular adminstration.The mode that combination treatment can comprise the interrupted circulation of rest period is carried out, so that the health of patient has an opportunity to recover from any still unforeseen side effect.Medicine also can be formulated into together, so that single administration sends this two kinds of medicines.
the preparation of pharmaceutical composition
The administration of often kind of medicine of combination can be undertaken by any applicable mode, make with other combination of components after, the concentration of medicine can improve the situation of patient (it can such as be determined the impact that the expression of PMP22 raises according to after arrival peripheral nervous in vitro).
Although they as pure chemical substance administration, can provide as pharmaceutical composition under preferable case, in this article also referred to as pharmaceutical preparation by the active component in combination.Possible compositions comprises the compositions being suitable for oral, rectum, surface (comprising transdermal, cheek and Sublingual) or parenteral (comprising subcutaneous, intramuscular, intravenous and intradermal) administration.
More generally, these pharmaceutical preparatioies are with the form of " the patient's bag " containing multiple dosage unit, or other being placed on individual packaging for difference being treated the measurement unit medicament used in period, being generally the mode of administration in blister package, open patient.Patient's bag and pharmacists are compared with packing greatly the conventional prescriptions medicine of the medicine supply separating patient pharmaceutical supply product, and its advantage is that patient always can obtain the package insert comprised in patient's bag, and it does not generally have in conventional prescriptions medicine.Comprise package insert to be shown and can to improve the compliance of patient to doctor's instruction.Therefore, the present invention also comprises aforesaid pharmaceutical preparation herein and the combination of packaging material being suitable for described preparation.In such patient bag, for the expection using method of the preparation of combined therapy, from description, equipment, supply, indication and/or other means helping use preparation to be the most compatibly used for the treatment of, can infer.Such measure makes patient wrap to be particularly suitable for and can transform the treatment that is applicable to carry out with combination of the present invention.
Medicine can any applicable amount be included in any applicable carrier mass, and can composition total weight weighing scale 1-99% amount exist.Compositions can be provided in the dosage form being suitable for oral, parenteral (such as intravenous, intramuscular), rectum, skin, nose, vagina, suction, skin (paster) or ocular administration approach.Therefore, compositions can take such as tablet, capsule, pill, powder, granule, suspension, emulsion, solution, gel to comprise hydrogel, paste, ointment, ointment, plaster, immersion, osmotic delivery device, suppository, enema, injection, implant, spray or aerocolloidal form.
Pharmaceutical composition can medicinal practice conveniently carry out preparing (see such as " Remington: pharmaceutical science with put into practice " (the 20th edition) (Remington:The Science and Practice of Pharmacy (20th ed.)), A.R.Gennaro edits, Lippincott Williams & Wilkins, 2000, and " pharmaceutical technology encyclopaedia " (Encyclopedia of Pharmaceutical Technology), J.Swarbrick and J.C.Boylan edits, 1988-1999, Marcel Dekker, New York).
Pharmaceutical composition of the present invention can be mixed with and substantially any predetermined time or period discharge active medicine at once or upon administration upon administration.
Controlled release formulation comprises the preparation that (i) produces the drug level of substantial constant in vivo in long period section; (ii) after predetermined time delay, in long period section, produce the preparation of the drug level of substantial constant in vivo; (iii) by maintaining the effective levels of drugs of relative constancy in body and minimizing relevant undesired side effect of fluctuating with the plasma concentration of active drug substance at the same time, pharmaceutically-active preparation is maintained within a predetermined period of time; (iv) by such as controlled-release composition space to be placed near illing tissue or organ or wherein, to make the preparation that drug effect localizes; And (v) delivers drugs into particular target cell type by using carrier or chemical derivative and makes the preparation of drug effect orientation.
Medicine is with the form administration of controlled release formulation, particularly preferred in situations, in such cases, have (i) narrow therapeutic index (difference namely produced between the plasma concentration of harmful side effect or toxic reaction and the plasma concentration producing therapeutic effect is little drug regimen; In general, therapeutic index TI is defined as the ratio of intermediate value fatal dose (LD50) and intermediate value effective dose (ED50)); (ii) narrow in the gastrointestinal tract absorbing window; Or the biological half life that (iii) is very short, make frequently medication blood plasma level must to be maintained treatment level within one day.
The controlled release that wherein rate of release exceedes discussed drug metabolism speed can be obtained according to any one in multiple strategy.Controlled release by compatibly selecting various different formulation parameters and composition, can comprise such as various dissimilar controlled-release composition and coating, obtaining.Therefore, the pharmaceutical composition (single or multiple unitary tablet or capsule composition, oil solution, suspension, emulsion, microcapsule, microsphere, nanoparticle, paster and liposome) of medicine is discharged after medicine being become administration with the excipient be applicable in a controlled manner.
The solid dosage forms orally used
The tablet of the mixture containing active component and the pharmaceutically acceptable excipient of avirulence is comprised for oral preparation.These excipient can be such as inert diluent or filler (such as sucrose, micro-crystalline cellulose, starch comprise potato starch, calcium carbonate, sodium chloride, calcium phosphate, calcium sulfate or sodium phosphate); Granulating and disintegrating agent (such as cellulose derivative comprises micro-crystalline cellulose, starch comprises potato starch, cross-linking sodium carboxymethyl cellulose, alginate or alginic acid); Adhesive (such as Radix Acaciae senegalis, alginic acid, sodium alginate, gelatin, starch, pre-gelatinized starch, micro-crystalline cellulose, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl emthylcellulose, ethyl cellulose, polyvinylpyrrolidone or Polyethylene Glycol); And lubricant, fluidizer and anti-binder (such as stearic acid, silicon dioxide or Pulvis Talci).Other pharmaceutically acceptable excipient can be coloring agent, flavoring agent, plasticizer, wetting agent, buffer agent etc.
Tablet can be without coating, or they can pass through known technology optionally coating, to postpone disintegrate in the gastrointestinal tract and absorption, provides lasting effect in a long time thus.Coating can be suitable for predetermined pattern release active drug substance (such as to obtain controlled release formulation), or it can be suitable for not discharging active drug substance (enteric coating) before by stomach.Coating can be sweet tablet, film coating is (such as based on hydroxypropyl emthylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic copolymer, Polyethylene Glycol and/or polyvinylpyrrolidone) or enteric coating (such as based on methacrylic acid copolymer, cellulose acetate phthalate, Hydroxypropyl Methylcellulose Phathalate, HPMCAS, Opaseal, Lac and/or ethyl cellulose).Material such as glyceryl monostearate or distearin can be postponed service time.
Solid tablet compositions can comprise the coating being suitable for protection group compound and avoiding undesired chemical change (chemical degradation before such as active drug substance release).Coating can be applied in solid dosage forms according to the similar mode described in " pharmaceutical technology encyclopedia " (Encyclopedia of Pharmaceutical Technology).
Medicine can mix in tablets, maybe can separate.Such as, the first pharmaceutical pack is contained in tablet inside, and the second medicine, in outside, makes the second medicine of considerable part discharge before the first drug release.
Preparation for orally using also can show as chewable tablet or as hard gelatine capsule, wherein active component mixes with inert solid diluent (such as potato starch, micro-crystalline cellulose, calcium carbonate, calcium phosphate or Kaolin), or as soft gelatin capsule, wherein active component and water or oily medium such as liquid paraffin or mixed with olive oil.The composition mentioned under Tablet and Capsula above powder and granule can use, prepares in a usual manner.
The controlled-release composition orally used, can be built into and such as discharge active medicine by the dissolving and/or diffusion that control active drug substance.
Dissolve or diffusion-controlled release, suitable coating can be carried out by the tablet of medicine, capsule, spherolite or granular preparation, or by medicine is mixed applicable substrate to realize.Controlled release coating can comprise one or more coating substances above-mentioned, and/or such as Lac, Cera Flava, glycowax, castor wax, Brazil wax, stearyl alcohol, glyceryl monostearate, distearin, palmitostearate, ethyl cellulose, acrylic resin, dl-polylactic acid, cellulose acetate-butyrate, polrvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylates, methyl methacrylate, 2-hydroxyl-metacrylate, methacrylate hydrogels, 1, 3-butanediol, methacrylic acid glycol ester, and/or Polyethylene Glycol.In controlled release matrix dosage form, host material also can comprise such as aqueous methylcellulose, Brazil wax and stearyl alcohol, acrylate copolymer 934, silicone, glyceryl tristearate, acrylic acid methyl ester .-methyl methacrylate, polrvinyl chloride, polyethylene and/or halo fluorohydrocarbon.
The controlled-release composition of one or more medicines containing claimed combination, also can take the form (namely tablet or capsule are after oral administration, the floating certain hour section at the top of stomach inclusions) of floating tablet or capsule.The floating tablet preparation of medicine, can by preparing the mixture granulating of the hydrogel of medicine and excipient and 20-75%w/w such as hydroxyethyl-cellulose, hydroxypropyl cellulose or hydroxypropyl emthylcellulose.Then the particles compress of acquisition can be become tablet.When contacting with gastric juice, tablet defines fluid-tight gel blockage layer substantially around its surface.This gel blockage layer take part in and density is maintained less than 1, thus makes tablet keep swimming in gastric juice.
The liquid of oral administration
Be adapted to pass through add water to prepare aqueous suspension powder, dispersible powder or granule, be the dosage form easily for oral administration.Preparation as suspension provides the mixture of active component and dispersion or wetting agent, suspending agent and one or more antiseptic.The suspending agent be applicable to is such as sodium carboxymethyl cellulose, methylcellulose, sodium alginate etc.
Parenteral composition
Pharmaceutical composition also can by with dosage form, dosage form injection, infusion or implantation (intravenous, intramuscular, subcutaneous etc.), or pass through the implant of applicable delivery apparatus or the nontoxic pharmaceutically suitable carrier containing routine and adjuvant, carry out parenteral.The preparation of these compositionss and preparation for field of pharmaceutical preparations professional be known.
The compositions used for parenteral can provide (such as in unit dose ampoule) by unit dosage forms, or is provided in the bottle containing several parts of medicaments, wherein can add applicable antiseptic (vide infra).Compositions can take solution, suspension, emulsion, infusion device or the form of delivery apparatus for implanting, or it can provide by dry powder, can before use with water or the another kind of medium reconstruct be applicable to.Except active medicine, compositions can comprise applicable parenteral acceptable carrier and/or excipient.Active medicine can be incorporated in microsphere, microcapsule, nanoparticle, liposome etc. for controlled release.Compositions can comprise suspending agent, solubilizing agent, stabilizing agent, pH adjusting agent and/or dispersant.
Pharmaceutical composition of the present invention can adopt the form being suitable for aseptic injection.In order to prepare such compositions, applicable active medicine is dissolved or suspended in the acceptable liquid medium of parenteral.Operablely accepting medium and solvent can comprise water, the water adjusting to applicable pH by adding appropriate hydrochloric acid, sodium hydroxide or applicable buffer agent, 1,3 butylene glycol, Ringer ' s solution and isotonic sodium chlorrde solution.Aqueous formulation also can comprise one or more antiseptic (such as methyl parahydroxybenzoate, ethyl ester or n-propyl).Medicine on a small quantity or a when being slightly dissolved in water, can add dissolution enhancers or solubilizing agent, or solvent can only can comprise the propylene glycol etc. of 10-60% w/w wherein.
The parenteral composition of controlled release can take the form of aqueous suspension, microsphere, microcapsule, magnetic microsphere, oil solution, oil suspension or emulsion.Alternatively, can be incorporated into can in biocompatible carrier, liposome, nanoparticle, implant or infusion device for active medicine.Material for the preparation of microsphere and/or microcapsule be such as biodegradable/can the polymer of bioerosion, such as PLGA, poly-(isobutyl cyanoacrylate base ester), poly-(2-hydroxyethyl-L-glutaminate).Operable when preparing controlled release parenteral administration can physiologically acceptable carrier be saccharide (such as glucosan), albumen (such as albumin), lipoprotein or antibody.The material used in implant can be not biodegradable (such as poly-dimethyl sulfoxine) or biodegradable (such as (polycaprolactone), poly-(hydroxyacetic acid) or poly-(ortho esters)).
Rectal compositions
Use for rectum, the dosage form being suitable for compositions comprises suppository (Emulsion or outstanding agent type) and rectum gelatin capsule (solution or suspension).In typical suppository formulations, active medicine mixes from pharmaceutically acceptable suppository base such as cocoa butter, esterified fatty acid, glycerinated gelatin and the various different water solublity or dispersible substrate such as Polyethylene Glycol be applicable to.Various different additive, reinforcing agent or surfactant can be mixed.
Percutaneous and surface composition
Pharmaceutical composition also can in the administration of skin upper surface, at dosage form or the preparation containing conventional nontoxic pharmaceutically suitable carrier and excipient, comprise percutaneous absorbtion in microsphere and liposome.Preparation comprises cream, ointment, lotion, liniment, gel, hydrogel, solution, suspension, pastes agent, spray, paste, plaster, and the transdermal drug delivery systems of other kinds.Pharmaceutically suitable carrier or excipient can comprise emulsifying agent, antioxidant, buffer agent, antiseptic, wetting agent, penetration enhancer, chelating agen, gelatinizing agent, ointment base, spice and Derma-Guard.
Emulsifying agent can be naturally occurring natural gum (such as Radix Acaciae senegalis or adragant).
Antiseptic, wetting agent, penetration enhancer can be metagin such as methyl parahydroxybenzoate or propyl ester, and benzalkonium chloride, glycerol, propylene glycol, carbamide etc.
Above-described for be also used at the pharmaceutical composition of skin upper surface administration on body part to be treated or near surface administration.Compositions goes for direct applying, or is applied by special drug delivery device such as dressing or optional plaster, pad, sponge, band or other forms of applicable flexible material.
therapeutic dose and persistent period
Should be realized that, the medicine in combination can in identical or different pharmaceutical preparation simultaneously or administration in succession.If administration in succession, the delay when the second (or additional) delivery of active ingredients should not make the benefit of the net effect of active ingredient combinations lose.For the combination of this description, minimum requirements is that combination should be expected the benefit of the net effect of active ingredient combinations carried out Combination application.The intended application of combination and/or can contribute to using other means of combination of the present invention to infer by equipment, supply, indication.
The medicine as theme of the present invention for the treatment of effective dose can make for preparing medicament together, described medicament can be used for the effect reducing PMP22 gene expression increase, recover normal myelin to be formed and neural-integrity, stop or reduce the risk that CMT disease occurs, once CMT disease become clinical obviously after stop or slow down its development, and stop or reduce the risk of first or follow-up generation of neuropathic event.
Although active medicine of the present invention can divided doses, such as every day 2 or 3 times, but often kind of medicine single-dose every day in combination is preferred, wherein all medicines are most preferred at single medicine compositions (unit dosage forms) single-dose middle every day.
Administration can be arrived several times once a day, continues several days to several years, and it is lifelong even can to continue patient.In most of the cases, will to rectificate phase or at least periodically repeatedly long term administration.
● term " unit dosage forms " refers to and is suitable as the physically separated unit (such as capsule, tablet or the injector syringe that install) of single dose for human subjects, and each unit contains the active material of the scheduled volume that can produce required therapeutic effect as calculated and required pharmaceutical carrier.
The amount being preferred for often kind of medicine in the combination of unit dose will depend on several factor, comprise medication, the body weight of patient and age, the seriousness of neuropathic damage that caused by CMT disease, or the risk of potential side effect in view of the general health of people to be treated.
In addition, about pharmacogenomics (genotype is on the effectiveness situation of the impact of pharmacokinetics, pharmacodynamics or the medicine) information of concrete patient, the dosage of use can be affected.
Except may higher dosage be needed when tackling special nocuity CMT disease, or should select when treating child comparatively outside low dosage, the preferred dose of often kind of medicine in combination, generally by within not exceeding the dosage range that is generally long-term maintenance treatment institute prescription or be proved to be safe dosage range in large-scale 3 phases clinical research.
Such as,
● for rapamycin, every day about 1, to about 100 μ g/kg, is typically 1 to 50 μ g/kg, such as, between 5 to 30 μ g/kg/ days.
● for mifepristone, every day about 1, to about 300 μ g/kg, is typically 10 to 200 μ g/kg, such as, between 10 to 80 μ g/kg/ days.
● for naltrexone, every day about 1, to about 100 μ g/kg, is typically 1 to 50 μ g/Kg, such as, between 1 to 20 μ g/kg/ days.
● for pilocarpine, every day about 1, to about 100 μ g/kg, is typically 1 to 50 μ g/Kg, such as, between 1 to 20 μ g/kg/ days.
● for baclofen, every day about 1, to about 300 μ g/kg, is typically 10 to 200 μ g/kg, such as, between 20 to 100 μ g/kg/ days.
● for thiamazole, every day about 1, to about 100 μ g/kg, is typically 1 to 50 μ g/kg, such as 1 to 20 μ g/kg/ days.
Most preferred dosage should be equivalent to 1% of the amount being generally long-term maintenance treatment institute prescription until 10%.
Should be appreciated that, the medication amount of actual administration is determined by doctor according to relevant situation, and described situation comprises disease to be treated, concrete compositions to be administered, age of individual patient, body weight and response, the seriousness of patients symptomatic and selected route of administration.Therefore, above-mentioned dosage range is intended to for telling about of this paper provides general guidance and support, and does not intend to limit the scope of the invention.
Give the following examples, its object is to illustrate instead of restriction.
Embodiment
A. the preparation of drug regimen
Prepare agents combination:
B. experiment in vitro
1. express algoscopy with the PMP22 on the Schwann cell of mixture 1-6 process
1.1 cell culture
1.1.1: commercially available rat primary Schwann cell
The bottle of rat Schwann cell (SC) primary culture (Sciencell # R1700) is thawed, with 10000 cell/cm 2density be seeded in the pre-coated 75cm of polylysine 2in " Sciencell Schwann cell culture medium " in culture bottle (the minimal medium #R1701 from Sciencell).Culture medium is grown enriching substance (3H Biomedical AB #1701-1752), 1% gentamycin (Sigma #G1397) and 10 μMs of forskolin (Sigma #F6886) and is formed by minimal medium, 5% hyclone (3H-Biomedical AB#1701-0025), 1% Schwann cell, to promote their propagation.
After reaching symphysis (4 to 10 days, depend on cell batch), eluriate by gentle agitation or by thy1.1 immunity, make SC be separated purification Schwann cell with adhesion fibroblast, to produce at least 95% pure culture.Then SC is counted (trypan blue method), and be inoculated into the 75cm that polylysine wraps quilt in advance 2in same SC culture medium in culture bottle.When symphysis, cell is washed, by trypsin treatment (from the 1x diluent of the trypsin-EDTA of Invitrogen #1540054, dilute in the PBS of not calcic and magnesium), counting, be plated in the Sciencell Schwann cell culture medium in 12 orifice plates (140000 cells/well), this culture medium is also containing 5%FBS, 1% Growth of Cells enriching substance (CGS), 40 μ g/ml gentamycins and 4 μMs of forskolin.
1.1.2 the rat primary Schwann cell customized
Primary Schwann cell culture (SC) is set up from Sprague-Dawley neonate rat (between P0 to P2) sciatic nerve.All neonate rats are put to death and cultivates in blood at Pi Shi and be separated.Dissect and aseptically carry out.
From rear solid end and lower torso removing skin of back.Isolate sciatic nerve and transfer in the culture dish containing ice-cold Leibovitz (L15, Invitogen #11415), being added with 1% penicillin/streptomycin solution in Leibovitz and (being respectively 50UI/ml and 50 μ g/ml; Invitrogen #15070) and 1% bovine serum albumin (BSA, Sigma A6003).Two of every rat nerves are all transferred in the 15ml test tube containing ice-cold L15.Then remove L15 culture medium, replace with the 2.4ml DMEM (Invitrogen #21969035) containing 10mg/ml collagenase (Sigma #A6003).By nerve in this culture medium at 37 DEG C incubation 30 minutes.Then culture medium is removed, by the trypsin 10% trypsin-EDTA 10x diluted with the PBS (Invitrogen #2007-03) of not calcic and magnesium, Invitrogen #15400054) 37 DEG C of process 20 minutes, two nerves are dissociated.Cessation reaction is carried out by the DMEM added containing II level DNase I (0.1mg/ml, Roche diagnostic#104159) and hyclone (FCS 10%, Invitrogen #10270).Cell suspension 10ml pipet is ground loose, is collected in (Swinnex 13mm defecator, Millipore use 20 μm of nylon screen filter membranes, Fisher) in 50ml pipe by filter.By cell suspension room temperature (RT) with 350g centrifugal 10 minutes, and precipitation is suspended in the DMEM containing 10%FCS and 1% penicillin/streptomycin.Cell is counted (trypan blue method), and with 5.10 5to 10 6the density of individual cell/plate is inoculated in Falcon 100mm Primaria tissue culturing plate.
In cultivation after 1 day, culture medium DMEM, 10% FCS, 1% penicillin/streptomycin and 10 μMs of cytosine b-D-arabinofuranosidase (Sigma #C1768) are changed.After 48 hours, removing culture medium, washs 3 times by cell DMEM.Then add SC growth medium, it is made up of DMEM, 10%FCS, 1% penicillin/streptomycin, 2 μMs of forskolin (Sigma #F6886), 10 μ g/ml cattle pituitary extract (PEX, Invitrogen #13028).Every 2-3 days replaced medium.
In cultivation after 8 days (4 to 10 days, depend on cell batch), Schwann cell reaches symphysis, carries out purification by containing a large amount of fibroblastic culture polluted by thy1.1 immunity elutriation method.After purification, by cell with 10000 cell/cm 2density suspension wrap the 75cm of quilt in advance at polylysine 2in growth medium in culture bottle.Once they reach symphysis, cell is cleaned, with trypsin treatment (trypsin-EDTA), counting, and be plated on (100000 cells/well) in 12 orifice plates.
1.1.3 medicine incubation
By plating cells after 12 orifice plates, culture medium is replaced by defined medium, this culture medium contains DMEM-F12 mixture (Invitrogen #21331020), and is supplemented with 1%N2 supplement (Invitrogen #17502), 1%L-glutamine (Invitrogen #25030024), 2.5%FBS (Sciencell #0025), 0.02 μ g/ml corticosterone (Sigma #C2505), 4 μMs of forskolin and 50 μ g/ml gentamycins.In this culture medium, do not add somatomedin, to promote that SC breaks up.
After 24 hours, this culture medium is replaced with defined medium (DMEM-F12), it is supplemented with 1% Insulin-Transferrin-selenium-X (ITS, Invitrogen #51300), 16 μ g/ml butanediamine (Sigma #P5780), 0.02 μ g/ml corticosterone and 50 μ g/ml gentamycins.In this step, neither there is progesterone in culture medium and also there is not forskolin.
After 1 day, by Schwann cell medicine combination of stimulation 24 hours (3 hole/conditions).Often kind of compound fresh preparation before being added in cell culture medium.
Medicine is added to by the defined medium that DMEM-F12 is formed, 1% Insulin-Transferrin-selenium-X (ITS, Invitrogen #51300), 16 μ g/ml butanediamine, 0.02 μ g/ml corticosterone, 10nM progesterone and 50 μ g/ml gentamycins are supplemented with in described culture medium.In medicine irritation process, there is not forskolin, avoid the sweet cyclase of acid of gland saturated.
1.2. purification Schwann cell is eluriated by Thy1.1 immunity
In order to prevent the pollution of fibroblast cell cultures, use clone Thy1.1 (ATCCTIB-103 tM) immune elutriation scheme carried out purification to Schwann cell.
The pre-coated 100mm antibacterial petri diss of antibody is prepared as follows: these plates PBS is washed 3 times, spends the night 4 DEG C of process with the 50mM Tris HCl solution of the 20ml pH 9.5 containing 10 μ g/ml goat anti-mouse IgM MU antibody (Jackson ImmunoResearch #115-005-020); Then 3 times are washed with PBS, with containing 0.02%BSA and the PBS solution of supernatant containing Thy1.1 IgM antibody that obtains from T1 1D7e2 Hybridoma culture (ATCC #TIB-103), room temperature treatment 2 hours.Finally, plate PBS is washed 3 times, then adds cell suspension.
SC trypsin EDTA unclamps.Once most cells enters suspension, use in DMEM-10%FBS immediately and trypsin, and by cell centrifugation.By the precipitation of cell of loosening with every ml 0.66x10 6the density Eddy diffusion of individual cell (maximum) contains in the culture medium of 0.02%BSA at 15ml, and transfers to (about 6,600,000 cells/10ml/100mm culture dish) in petri diss.
Cell suspension to be wrapped in the petri diss of quilt at 37 DEG C incubation at Thy 1.1 45 minutes, within every 15 minutes, stir gently to prevent non-specific binding.The fibroblast that major part expresses Thy1.1 attaches on culture dish.At the end of incubation, recovery cell suspension is also centrifugal.This cell suspension is in theory only containing Schwann cell.By cell centrifugation, by cell precipitation with 16000 cell/cm 2density suspension at the T75cm of polylysine process 2containing in the growth medium of 10 μMs of forskolin in culture bottle.
1.3 quantitative Reverse Transcriptase polymerase chain reactions (Q-RT-PCR)
Quantitative RT-PCR is used to comparative drug stimulates rear PMP22 mRNA relative to the level of the house keeper's ribosome L13A mRNA in rat Schwann cell primary culture.
After with cold aseptic PBS cleaning, from SC, use the total serum IgE of each cell sample of Qiagen RNeasy mini kit (Qiagen #74004) Isolation and purification.Use 1 μ l RNA sample, by Nanodrop spectrophotometer, nucleic acid is carried out quantitatively.RNA integrity is determined by BioAnalyzer (Agilent) device.
According to standard scheme, RNA reverse transcription is become cDNA.CDNA template for pcr amplification is synthesized from 200ng total serum IgE, employ SuperScript II reverse transcription (Invitrogen #18064-014), when there is oligomerization (dT) 42 DEG C of reactions 60 minutes, final volume is 20 μ l.
Use system (Roche Molecular Systems Inc.) carries out pcr amplification to cDNA.Before for pcr amplification, by each cDNA diluted sample 5 times.2.5 these cDNA of μ l are joined (final volume 10 μ l) in PCR reaction solution.Carried out preliminary experiment to guarantee to carry out in the exponential phase of the amplification procedure of two kinds of sequences quantitatively, and the expression of reference gene is consistent under different condition of culture.
PCR reaction is undertaken (increased 148-bp) by the 500nM forward primer 5-GGAAACGCGAATGAGGC-3 (SEQ ID NO:1) of P of Rats MP22 (NM_017037) and the amplification of 500nM reverse primer 5-GTTCTGTTTGGTTTGGCTT-3 (SEQ ID NO:2).By using 500nM forward primer 5-CTGCCCTCAAGGTTGTG-3 (SEQ ID NO:3) and 500nM reverse primer 5-CTTCTTCTTCCGGTAATGGAT-3 (SEQ ID NO:4), increased abreast the fragment of 152-bp of RPL13A ribosome (NM_173340) RNA in the reaction separated, for being normalized result.
We used FRET chemistry to carry out RT-Q-PCR analysis.FRET probe is made up of, at their 3 ' end donor fluorophore dyestuff (fluorescein) labelling 0.3 μM of Pmp22-FL-5-GCTCTGAGCGTGCATAGGGTAC (SEQ ID NO:5) or Rpl13A-FL-5-TCGGGTGGAAGTACCAGCC (SEQ ID NO:6).0.15 μM of Red640 probe is defined as follows: Pmp22-red-5 '-AGGGAGGGAGGAAGGAAACCAGAAA (SEQ ID NO:7) or Rpl13A-red-5 '-TGACAGCTACTCTGGAGGAGAAACGGAA (SEQ ID NO:8), rolls into a ball dyestuff (rhodamine Red 640) labelling in their 5 ' end acceptor fluorescence.
Each PCR reaction is comprise 2.5 μ l cDNA templates in the main mixture test kit (Roche#04-887301001) of 10 μ l at cumulative volume.
Employ following PCR condition: 95 DEG C 10 seconds, 63 DEG C 10 seconds, 72 DEG C of 12 seconds and 40 DEG C 30 seconds (40 amplification cycles).The relative level of PMP22 gene expression is calculated by the ratio measured between the product that produces from target gene PMP22 and endogenous internal controls RPL 13A.
1.4 carry out PMP22 protein expression analysis by flow cytometry (FACS)
With medicine incubation after 8 hours, 24 hours and 48 hours, reclaim supernatant, centrifugal and freezing.Use trypsin-EDTA partition SC.Once most cells enters suspension, use in the DMEM containing 10%FCS immediately and trypsin.
To reclaim containing the supernatant of cell and centrifugal.Cell precipitation is transferred in micro tube, clean once in PBS, use particular solution (AbCys #Reagent A BUF09B) to fix.After 10 minutes, by cells rinsed with PBS once, and remain on 4 DEG C.
Cell fixes latter 5 days, and use scheme below, the cellular preparations different to all incubative times carries out labelling.
By cell with 7000rpm centrifugal 5 minutes, precipitation is suspended in permeabilized solution (AbCys#Reagent B BUF09B), with PMP22 first antibody (Abcam #ab61220,1/50) room temperature labelling 1 hour.Then by cell with 7000rpm centrifugal 5 minutes, cell precipitation is washed once in PBS.Add and the second antibody of Alexa Fluor 488 coupling (goat anti-rabbit igg, Molecular Probes #A11008,1/100), room temperature reaction 1 hour.Then by cell with 7000rpm centrifugal 5 minutes, cell precipitation is washed once in PBS.Add and the 3rd antibody of Alexa Fluor 488 coupling (chicken anti goat igg, Molecular Probes#A21467,1/100), incubation at room temperature 1 hour, to increase labelling.Then cell is washed once in PBS.Perform not containing the contrast (unlabeled cells) of any antibody, to determine the level of autofluorescence and to adjust the sensitivity of light multiplexer.Perform use second and the 3rd antibody but do not use the contrast of first antibody, to assess the non-specific binding of antibody.
Data acquisition and analysis use FACS array cell counter and FACS array software (Becton Dickinson) to carry out on 5000 cells.Analyze the forward scatter (FSC) relevant to cell volume (size) and depend on the lateral scattering of internal cellular complexity (granularity).For the expression of PMP22, analyze in total cell, and calculate the percent of positive cell.Positive cell is the cell of fluorescence intensity higher than the contrast of use second antibody.
In order to carry out quantitatively to SC quantity, anti-S100 protein antibodies is used to analyze cell in control medium.
Cell is prepared according to following scheme: dyeed 1 hour in room temperature with anti-S 100 protein antibodies (Dako#S0311,1/100) by Schwann cell.This antibody carries out labelling according to the above-described scheme for PMP22 immunostaining, but not with the 3rd antibody incubation.
1.5. medicine incubation and activity
By medicine with incubation in above-described identical defined medium 24 hours or 48 hours (3 hole/conditions), there is not forskolin in described culture medium saturated with what avoid adenyl cyclase to stimulate, but there is 10nM progesterone.With medicine incubation after, reclaim supernatant, analyze freezing for Schwann cell for RT-Q-PCR.
These General description of experiments in Table 1.
Table 1
Combination PMP22 expresses
Mixture 1 Lower
Mixture 2 Lower
Mixture 3 Lower
Mixture 4 Lower
Mixture 5 Lower
Mixture 6 Lower
2. in the co-culture model of CMT, assess the cooperative effect of the compound in mixture 7
Use co-culture model as the external model of CMT1A.The Schwann cell of the dorsal root ganglion (DRG) that this myelin formation model is separated with from male PMP22 transgenic rat (TG) by the sensory neuron of Dual culture is formed.
The object of this research is assessment 3 kinds of test compounds (+/-baclofen, naltrexone and Sorbitol) and mixture 7 (mixture of these the three kinds of medicines) impact on myelin forming process.The impact that 3 kinds of test compounds and composition thereof are formed myelin, is evaluated by the expression of assessment myelin basic protein (MBP) under ascorbic acid exists.
2.1 materials and methods
The gestation pregnant female rat of 15 days is killed by cervical dislocation method.From uterus, take out embryo, they are in identical Embryonic Stages.
2.1.1 gene type
Each embryo's head is got one piece of (3mm 3) be placed in 2ml test tube without DNase.SYBR Green extract-N-Amp is used to organize PCR kit (Sigma, reference number XNATG-1KT) to extract DNA.120 μ l are extracted solution (Sigma test kit, reference number XNATG-1KT) be placed on every block embryo head.By head incubation at room temperature 10 minutes.At the end of this incubation, by head in extraction solution at 95 DEG C incubation 5 minutes.After this last incubation, add 100 μ l neutralization solutions immediately, use aseptic ultra-pure water (Biosolve, reference number: 91589) by often kind of DNA extraction thing with 1/40 dilution, and be stored in+4 DEG C until use.Between DRG separation period, the test kit main mixture of Fast SYBR Green (Applied Biosystem, 4385612) is used to carry out the gene type of female (F) and male (M) embryo.The sex of each embryo uses male sry gene to determine.SRY primer provides (SRY-F (SEQ ID NO:9): 5 '-GAGAGAGGCACAAGTTGGC-3 ' by Pharnext; SRY-R (SEQ ID NO:10): 5 '-GCCTCCTGGAAAAAGGGCC-3 ').By SRY primer at aseptic ultra-pure water (Biosolve, reference number: be diluted to 3 μMs 91589).Mixture for PCR uses ultra-pure water (each sample 4 μ l), 3 μMs of primers (each sample 2 μ l) and main mixture (each sample 10 μ l) to prepare.In 96 hole PCR plate, in each hole, place 16 μ l PCR mixture.Arrange according to schedule and add the DNA that 4 μ l often plant dilution.Use 7500 quick RT-PCR systems (Applied Biosystem) to run PCR, employ follow procedure:
Start: 95 DEG C-20 seconds
45 circulations: 95 DEG C-10 seconds, 65 DEG C-10 seconds, 72 DEG C-30 seconds (data acquisition).
Melting curve: 95 DEG C-15 seconds, 64 DEG C-1 minute, 90 DEG C-30 seconds (continuous data acquisition), 60 DEG C 15 seconds.Amplification figure and melting curve use 7500 softwares (Applied Biosystems) to analyze.
The result of each sample and negative control (ultra-pure water) and positive control (TG/ is male and WT/ is female) are compared, to draw a conclusion to the genotype of each embryo.
2.1.2 sensory neuron and Schwann cell coculture
Rat dorsal root ganglion is according to former Cosgaya etc., and 2002 and Rangaraju etc., the description of 2008 is cultivated.
By each embryo in the upper disposal of counting petri diss (diameter 35mm).Cut the head of embryo, be placed in the 1.5ml test tube without DNAase; Extract-N-Amp Tissue kit (Sigma Aldrich) is used to extract AND.Use the test kit main mixture of Fast SYBR Green (Applied Biosystem, 4385612) carry out gene type (male (M) and female (F), wild type and PMP22 genetically modified).This gene type carries out concurrently with being separated of dorsal root ganglion (DRG), at the end of separation, only to carry out the cultivation (DRG from Transgenic male) of a type.Collect the DRG of each embryo, be placed in ice-cold Leibovitz culture medium (L15, Invitrogen).At the end of separation, the DRG of TGM is merged, and by using trypsin trypsin EDTA at 37 DEG C, 0.05%; Invitrogen) process is loosened for 20 minutes.Cessation reaction is carried out by the DMEM added containing 10% hyclone (FBS) under DNAase I (Roche) existence.Suspension 10ml pipet is ground loose.Then by cell at room temperature with 350xg centrifugal 10 minutes.Cell precipitation after loosening is resuspended in containing 2%B27 (Invitrogen), 1% Pen .-Strep (Invitrogen), 1% L-glutaminate and 50ng/ml NGF (Sigma) neural minimal medium (Invitrogen) in.This culture medium is neuronal culture.Use Trypan Blue exclusion test (Sigma) to count living cells in Neubauer cell counter, and it is seeded in 96 orifice plates (Greiner) with polylysine process with the amount of 10000 cells in each hole.By plate in humidified incubator, at air (95%)-CO 2(5%) at maintaining 37 DEG C in atmosphere.Every other day change the standard neuronal culture of half.Culture is maintained 7 days in the neural minimal medium of standard, migrates in sensory neuron aixs cylinder to allow Schwann cell.At the 7th day, to culture medium supply supplement or the standard neuronal culture not augmenting 50 μ g/ml ascorbic acids, so that initial basement membrane is formed and myelin is formed.
2.1.3. medicine incubation
At the 7th day, following test compounds (alone or in combination) is added in the culture medium containing 50 μ g/ml ascorbic acids:
■ (RS)-baclofen
■ naltrexone
■ d-Sorbitol
The combination of ■ mixture 7=3 kind individuation compound
With following concentration, compound or compound combination are tested (table 2):
Table 2: the individual drugs of in vitro study expressed for MBP in TG DRG/SC coculture or the concentration of combination
By test compounds incubation 5 kinds of different times: 5,9,10,11 and 13 days.
Three parts that have carried out DRG (coming from TG male rat embryo) are separated and independently cultivate.These conditions are assessed, often kind of condition 6 holes under ascorbic acid exists.All test compounds with need prepare from storage solution with solution temporarily, be stored in-20 DEG C.The preparation in one week of this solution once.The supplement every other day changing half has the standard neuronal culture of test compounds and ascorbic acid (respectively with 1X concentration).
2.1.4 Staining Protocol
At incubation after 5,9,10,11 and 13 days, cell cold ethanol (95%) and acetic acid (5%) solution are fixed 10 minutes.Use the PBS containing 0.1% saponin and 10% lowlenthal serum by Cell permeabilization and blocking-up 15 minutes.Then by cell and the anti-myelin basic protein of myelinic Specific marker, i.e. polyclonal antibody (MBP) antibody (Sigma 118K0431) incubation.
This antibody Alexa Fluor 568 goat anti-rabbit IgG antibody and Alexa Fluor 488 goat anti-mouse IgG antibody (Molecular probe 687621,623962) detect.Neuronic core fluorescent marker (Hoechst solution, SIGMA ref B1155) labelling.
2.1.5. date processing
For each hole, InCell AnalyzerTM 1000 (GE Healthcare) is used to obtain 20 photos with 20x amplification.All images obtain under the same conditions.The total length analysis of marrow aixs cylinder is had to use Developer software (GE Healthcare) automatically to carry out.All values are expressed as meansigma methods +/-average standard error.Statistical analysis carries out at different conditions (after ANOVA, when permitted, carrying out Fisher ' s PLSD check).
2.2. result
The cooperative effect of the usefulness Chinese medicine of mixture 7
MBP expression observed the important cooperative effect being combined the medicine formed by mixture 7.In fact, (=cultivate the 17th day) on the 10th, the dosage 1 that being combined in of (RS)-baclofen, naltrexone and d-Sorbitol shows in Figure 1A and 2A and significantly increase MBP for 6 times and express.On the contrary, be used alone said medicine and there is no positive effect (Figure 1B-D and 2B-D) compared with the control.
At dosage 2,3,4,5 and 7 times incubations of mixture 7 after 10 days, also have recorded the active effects (Fig. 3 A) that MBP is expressed.
Using dosage 2-7, still observed this effect (Fig. 3 B) taking obvious bell shaped curve form at the 11st day.
c. the experiment in vivo in CMT animal model
We test the therapeutic effect of compound in rat model.
The Immature rat of two kinds of sexes is used to form experimental group respectively.Rat is assigned in group according to randomizing scheme according to body weight.In some experiments, randomization is carried out according to the performance of rat in bar test.Two kinds of sexes are represented by independently matched group, and they are quantitatively identical with processed group or larger.
Rat medicine long-term disposal---during 3 or 6 weeks, according to the bioavailability of often kind of medicine, undertaken by forcing feeding or using Alzet to permeate subcutaneous pump (DURECT Corporation Cupertino, CA) injection.In all experiment in vivo carried out, mixture 7 is by gavage administration.
Animal weighs weekly twice, so that according to the growth regulator amount of body weight.If have selected osmotic pumps to carry out process administration, calculate the dosage of medicine in the estimation average weight that the age of pump period (6 weeks) is estimated according to animal.If needed, applicable anesthesia scheme implantable pump again can be used.
performance testing
Every 3 or 4 weeks, performance testing is carried out to animal.Each test, is carried out the same time of one day by same research worker in same room; This concordance is maintained in whole experimentation.It is all blind that all process and genotype are determined for research worker.In whole research, mainly use " bar test (Bar test) " and " grip strength (Grip strength) " to assess behavior.Along with the growth of animal, the scheme of bar test can change (to avoid the deviation caused owing to such as learning).
The mensuration of grip strength allows to detect the nuance in grasping behavior, and it seems that grasping behavior to be made up of muscle strength, sensitivity state (tactile feel of such as pain may change the force measured), behavior component (" motivation ").Value between the limb of front and back is different, and greatly depends on the age of animal.
Grip strength thermometrically animal to be held in the strength on holding rod individually with its fore paw or rear solid end.Strength (Force Gauge FG-5000A) measured by the sthenometer put together with holding rod.Rat is caught by experimenter, makes it hold holding rod with its fore paw or with its rear solid end, and is pulled back gently by rat, until it decontrols holding rod.Record the power measured when animal release holding rod.
For every animal, carry out twice continuous print and measure the test of fore paw strength and the test of twice continuous print measurement rear solid end strength; Only record maximum score value (fore paw one, rear solid end one) (unit is N).
bar is tested
Bar test assessment rat holds the ability of fixed bar.The Pmp22 rat demonstrating Muscle weakness shows do very well (Sereda etc., 1996) in this experiment.Rat is placed, makes its four paws at the centre (shank diameter: 2.5cm of bar; Length: 50cm; Higher than desktop 30cm).Test is carried out continuously; In our experiment, the quantity of test and persistent period depend on animal batch.Variational introducing in this test is to determine the scheme being suitable for detecting movement defect in CMT rat best in experimentation.
Record the performance index of every first phase:
-on bar, keep the quantity of the test needed for 60 seconds (or for first first phase and the second phase 30 seconds).
-test the meansigma methods of time (namely falling the waiting time) of spending on bar and every first phase at every turn.Stopped in deadline, experimental arrangement that namely after 30 or 60 seconds, this phase terminates on bar rat, incomplete test will be gone back and be set as carrying out (such as deadline (30 seconds or 60 seconds), for the 8th batch, stop less than 10 seconds on bar in test 1,2 and 3, then in test 4 and 5, stopped the animal of 60 seconds, test 6 to 10 has been set as 60 seconds).
-the number of times that falls.
general health is assessed
The body weight of animal, outer aobvious sign (fur outward appearance, body gesture, gait, tremble) is monitored in whole experimentation.Service rating rating scale carries out record: 0=is normal, and 1=is abnormal.
gait
Every rat is observed 5 minutes in the new mouse cage (being of a size of 55x33x18cm) not having foreign material.The gait of rat uses 4 parameters to assess:
-0 point: normal gait (smoothness)
-1 point: abnormal gait (not smooth or rat is micro-lame)
-2 points: MD (rat is one leg in tow, and can be adjusted it also walk)
-3 points: serious anergy (rat its one or two rear solid end in tow, but can not adjust it/they).
surface test
Carriage has 30x50cm lucite plane, and it can with the angular slope of 0 ° (level) to 60 °.At the beginning, by every rat with head upwards position (head-up direction) be placed on inclined-plane, 25 ° of angles; Perform the test of being separated by for twice 1 minute.After 30 minutes, on inclined-plane, 35 ° of angles, then, on inclined-plane, 40 ° of angles, complete same experiment.During this period, rat is put back to cage.Clean inclined-plane after each test.
The performance of rat is by 4 different score value assessments:
-0 point: fricton-tight
-1 point: a little slip (or two pawls)
-2 points: moderate is slided (4 pawls), but does not slide into surface bottom
-3 points: rat slides into inclined-plane lowermost end always.
other tests
When applicable, electrochemistry assessment carried out to rat, histology measures, and pmp22 rna expression level in sciatic nerve is carried out quantitatively.
by quantitative RT-PCR, the pmp22 RNA in sciatic nerve is carried out quantitatively
According to manufacturer flow scheme (Qiagen-RNeasy fibrous tissue handbook) described in, use Qiazol (reference number 79306, Qiagen Gmbh, Germany), then RNeasy small volume of reagent box (reference number 74106 is used, Qiagen Gmbh, Germany) by single step purification method, be separated total serum IgE from left sciatic.Use DNA-free test kit (Qiagen-Rnase-free dnase set 1500 Kunits, reference number 1023460), by digesting removing DNA pollution with the DNase I without RNase.
Estimate RNA concentration by NanoDrop ND-1000, and carry out quality control testing by Agilent RNA 6000 nano chips on Agilent 2100Bioanalyzer.
Reverse transcription and PCR in real time: quantitative RT-PCR (RT-Q-PCR) carries out as follows: in 20-μ l reaction volume, use SuperScript tMiI reverse transcription (Invitrogen, Carlsbad, CA) and Oligo (dT) 12-18 (Invitrogen, Carlsbad, CA), carry out reverse transcription to 80 ng total serum IgE.
PCR in real time use rapid thermal cycles instrument system ( 480 II, 384 holes, Roche, Switzerland) perform.Amplification uses the primer concentration optimized between 130nM to 1 μM to carry out in 10 μ l cumulative volumes.Add to primer and template the main mixture of 480 SYBR Green I (2 × concentration, Roche, catalogue reference number 04 887 352 001).Comprise nucleotide, MgCL in the mixture 2, Taq archaeal dna polymerase and buffer.Amplification scheme comprise at first 95 DEG C of incubations 10 minutes to activate Taq archaeal dna polymerase, then 95 DEG C of degeneration 10s of 45 circulations, 60 DEG C of annealing 40s and 72 DEG C of extension 10s (being performed the detection of fluorescence-causing substance at the end of 72 DEG C of extended peroids by single acquisition mode) is carried out, finally carry out melting curve circulation, i.e. 95 DEG C of degeneration 5s, 63 DEG C of annealing 60s and 95 DEG C (heating rate is 0.11 DEG C/s from 63 DEG C to 95 DEG C, and continuous detecting fluorescence-causing substance).In order to confirm specific amplification, melting curve analysis is carried out to the PCR primer from each primer pair.Relative quantification is carried out in cross point (Cp value) according to each PCR primer.The fluorescence of the sample point be increased to higher than background fluorescence is called as the cross point (Cp) of sample.Brown rat (Rattus norvegicus) myelin protein Zero (MPZ) gene is used to be normalized (Sereda etc., 2006).The primer sequence (being synthesized by the Eurofins MWG Operon of Germany) analyzed for RT-Q-PCR is:
PMP22-forward: 5 '-TGTACCACATCCGCCTTGG-3 ' (SEQ ID NO:11) and
PMP22-is reverse: 5 '-GAGCTGGCAGAAGAACAGGAAC-3 ' (SEQ ID NO:12).
MPZ-forward: 5 '-TGTTGCTGCTGTTGCTCTTC-3 ' (SEQ ID NO:13) and
MPZ-is reverse: 5 '-TTGTGAAATTTCCCCTTCTCC-3 ' (SEQ ID NO:14).
result
Mixture 1 compositions improves bar test performance (Fig. 4) in whole handling procedure.
As shown in Figure 5, mixture 1 improves the gait score value of transgenic rat after process 3 and 6 weeks.
As shown in Figure 6, mixture 1 improves the performance of transgenic rat in surface test in process after 3,6,9 and 12 weeks.
Fig. 7 shows the positive-effect of mixture 2 to the gait score value of transgenic rat in 25,35 and 40 ° of surface tests.
Mixture 7 (dosage 3) significantly reduces the gene expression (Fig. 9) of pmp22RNA in the sciatic nerve of pmp22 transgenic rat.
(Figure 10) is improved with the performance of pmp22 rat in 35 ° of surface tests that mixture 7 (dosage 2 and dosage 3) processes.More particularly, 29 and 33% rat belong to performance good group, be 5% for TG placebo group in contrast to this, and 29 and 11% rat belong to performance bad group, be 60% for TG placebo group in contrast to this.For the TG rat processed with mixture 7-dosage 2, P-value (compared with TG placebo group) equals 0.0152, and for the TG rat processed with mixture 7-dosage 3, the p-value compared with TG placebo group equals 0.002.
Mixture 7-dosage 3 process significantly increase after 9 weeks pmp22 rat bar test in fall the waiting time (Figure 11): black dotted lines, p=4.56.10 -2, n=18.Also been observed TG placebo rat (melanin line, n=20) and WT placebo rat (grey pigmented line, p=3.82.10 -7, n=18) between significant difference.
Figure 12 shows the improvement of the grip strength of the pmp22 rat processed with mixture 7-dosage 3.
Figure 13 shows the remarkable relatedness between bar test waiting time (after process 9 weeks with mixture 7-dosage 3) and pmp22RNA expression.
Figure 14 show with mixture 7-dosage 3 process the bar after 9 weeks test the waiting time and the conduction velocity of Sensitive nerve (tail) between remarkable relatedness.
Similar results (see table 3) is obtained for other combinations.
Table 3
Combination PMP22 rat disease phenotype
Mixture 1 Improve
Mixture 2 Improve
Mixture 3 Improve
Mixture 4 Improve
Mixture 5 Improve
Mixture 6 Improve
These data show, and in vivo, combination of the present invention and therapeutic modality allow effectively to treat CMT.
d. the vivo effect in toxic neuropathy disease model
Drug treating or therapeutic modality carry out oral administration from the previous day (D16) of first time peritoneal injection 3mg/kg oxaliplatin the previous day (D-1) to the last test day.Belong to the animal daily distilled water (10ml/kg) of oxaliplatin processed group.Animal every day is the treatment tested of administration and distilled water in the morning, and oxaliplatin administration in the afternoon.
In test day (i.e. D1, D4, D10) period, administration process and distilled water after a test.For the test day (D4) comprising compound and medium administration and oxaliplatin injection, process and distilled water after a test, administration before oxaliplatin injection.From the animal only administration during test day (i.e. D1, D4, D10 and D17) of reference processed group.
By measuring the response to non-nocuity thermostimulation (acetone test) at D1 (after first time injection 3mg/kg oxaliplatin 24h (oxaliplatin acute effect)), D4, D10 (chronic effect of oxaliplatin) and the D17 residual effect of one week (oxaliplatin after processing is completed), assess crymodynia exception.
Test uses acetone test to carry out in 2 hours after reference substance administration.Reference material is oral 100mg/kg gabapentin (once a day x4 test day).
Acetone test
Acetone test is used to assess crymodynia abnormal.In this experiment, measure the waiting time (response time) of rear solid end retraction after applying an acetone to the sole of the foot face of two rear solid ends and (cold score value) is given a mark to respective strengths.
The response time to the cooling effect of acetone is measured in 20 seconds (deadline) after applying acetone.Also according to the grade of following 4 points, classification is carried out to the response of acetone: 0 (without response); 1 (snapback flicks pawl); 2 (bounce back for a long time or significantly flick pawl); 3 (repeat to flick pawl and with licking or stinging).
Rat is used to carry out 6 tests.For each experimental group, result is expressed as cumulative cold score value, summation ± SEM that its 6 score values being defined as every rat are combined.Minimum score value is 0 (arbitrary secondary all without response to 6 tests), and maximum possible score value is 18 (all repeating each time to flick and lick or sting pawl 6 tests).
gabapentinsource: Zhejiang Province, China chiral drug chemical company (Zhejiang Chiral Medicine Chemicals, China)
oxaliplatinsource: Sigma, France.
Result describes in fig. 8.They clearly demonstrate the neuropathic protectiveness effect that compositions of the present invention is induced oxaliplatin.
e. the vivo effect in ALS model
animal model
We select SOD1 g93Arat model (by generations such as Howland) simulates amyotrophic lateral sclerosis pathology.The SOD1 gene of this model process LAN sudden change in spinal cord, many brain regions and peripheral tissues.The outbreak of the motor neuron disease of this model is about 115 days time; It shows as hind leg abnormal gait.In several days, hindlimb paralysis rises.
experimental procedure
We pass through breeding stock SOD1 g93Arat and Sprague Dawley female rats are hybridized and are obtained colony.Use hSOD1 Auele Specific Primer, identify heterozygosis SOD1 by the polymerase chain reaction (PCR) of afterbody DNA g93Arat [1].Animal is maintained in the room with controlled illumination (in 0500-1900h illumination) and temperature (23 ± 1 DEG C), and allow freely close to food and water.Institute in this research performs according to animal care guidance standard in steps.
Carry out measured body weight weekly, behavior test starts when 60 age in days and lasts till terminal.From 5 week age, every day was processed by oral or subcutaneous way.
1. viewing test: general situation characterizes
Every rat is observed 5 minutes in the new mouse cage (size 55x33x18cm) not having foreign material.Have recorded 5 kinds of different parameters:
Gait
-0 point: normal gait (smoothness)
-1 point: abnormal gait (not smooth or rat is micro-lame)
-2 points: MD (rat is one leg in tow, and can be adjusted it also walk)
-3 points: serious anergy (rat its one or two rear solid end in tow, but can not adjust it/they.
Fur situation
-0 point: the sliding fur of clean and silk
-1 point: perpendicular hair or dirty fur
Tremble
-0 point: atremia
-1 point: tremble
Position
-0 point: normal
-1 point: abnormal (flattening or its back of the body of arching upward)
Rear solid end position
-0 point: normal
-1 point: stretch rear solid end
2. motion score value is tested: the sign of movement defect
This test assessment rat after being translated into either side in 30 seconds by ability (righting reflex) (Gale etc.) of himself normotopia.
Employ the nonparametric scoring system (Matsumoto etc., Thonhoff etc.) according to these standards below:
-0 point: rat can not in 30 seconds from either side by himself normotopia
-1 point: rat just can not in 30 seconds from certain side by himself normotopia
-2 points: rat can in 30 seconds from both sides by himself normotopia, but can not stand in cage; Some part of its always health in tow
-3 points: rat can in 30 seconds from both sides by himself normotopia, can not stand in cage, but some part of not health in tow
-4 points: rat can in 30 seconds from both sides by himself normotopia, can stand in cage, but there is visible functional defect
-5 points: rat can in 30 seconds from both sides by himself normotopia, can stand in cage, and there is no visible functional defect.
The terminal of disease is fixed on 0 point; Then by rat euthanasia.
3. surface test: the sign of movement defect
Carriage has 30x50cm lucite plane, and it can with the angular slope of 0 ° (level) to 60 °.At the beginning, by every rat with head upwards position (head-up direction) be placed on inclined-plane, 25 ° of angles; Perform the test of being separated by for twice 1 minute.After 30 minutes, on inclined-plane, 35 ° of angles, then, on inclined-plane, 40 ° of angles, complete same experiment.During this period, rat is put back to cage.Clean inclined-plane after each test.
The performance of rat is by 4 different score value assessments:
-0 point: fricton-tight
-1 point: a little slip (or two pawls)
-2 points: moderate is slided (4 pawls), but does not slide into surface bottom
-3 points: rat slides into inclined-plane lowermost end always.
4. metal gauze is tested: the sign of motor capacity under difficult situation
Metal gauze is placed to and contacts with chest at top at (with 70 ° of inclination angles) and contact with table edge in bottom.Every rat is placed in wire mesh bottom, and encourages it to climb by being placed in the chest at top by its brood newborn animal.Every rat is trained weekly once (3 tests).
The parameter of record is the waiting time arriving metal gauze top.
5. open ground is tested: autonomic activities capability representation
Autonomic activities ability is measured having in the lucite chest of 16 photocell beam along two axles being positioned at above bottom surface 1 and 5cm place (45x45x30cm, Acti-Track, BIOSEB, Lyon, France).
The spontaneous of every rat and exploration activity is assessed in 3 hours.
Record 4 parameters (total displacement, to stand number of times with hind leg, the percent in the time that the Distance geometry of center, open ground movement spends).
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Claims (5)

1. a compositions, it comprises baclofen, Sorbitol and naltrexone, their salt, enantiomer or raceme, for simultaneously, delivers medicine to mammalian object respectively or in succession, is used for the treatment of the toxic neuropathy that Sha-Ma-Tu is sick or drug-induced.
2. the compositions of claim 1, Sorbitol is wherein D-Sorbitol.
3. the compositions of claim 1 or 2, it comprises naltrexone, baclofen and D-Sorbitol.
4. the compositions of claim 1, it also comprises applicable medicinal excipient or carrier.
5. the compositions of claim 1 is manufacturing the application in the medicine being used for the treatment of the sick or drug-induced toxic neuropathy of Sha-Ma-Tu.
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